A neutrophil elastase inhibitor, sivelestat, ameliorates lung injury after hemorrhagic shock in rats

Hemorrhagic shock followed by resuscitation (HSR) causes neutrophil sequestration in the lung which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play a pivotal role in the pathogenesis of ALI. This study investigated whether sivelestat, a specific NE inhibitor, can attenuate ALI induced by HSR in rats. Male Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood so as to maintain a mean arterial blood pressure of 30+/-5 mm Hg for 60 min followed by resuscitation with the shed blood. HSR-treated animals received a bolus injection of sivelestat (10 mg/kg) intravenously at the start of resuscitation followed by continuous infusion for 60 min (10 mg/kg/h) during the resuscitation phase, or the vehicle. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, myeloperoxidase (MPO) activity, gene expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), DNA binding activity of nuclear factor (NF)-kappaB, and immunohistochemical analysis of intercellular adhesion molecule (ICAM)-1. HSR treatment induced lung injury, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, MPO activity, gene expression of TNF-alpha and iNOS, and DNA-binding activity of NF-kappaB, and enhanced expression of ICAM-1. In contrast, sivelestat treatment significantly ameliorated the HSR-induced lung injury, as judged by the marked improvement in all these indices. These results indicate that sivelestat attenuated HSR-induced lung injury at least in part through an inhibition of the inflammatory signaling pathway, in addition to the direct inhibitory effect on NE.     

Fas ligation triggers apoptosis in macrophages but not endothelial cells

The reticuloendothelial system includes macrophages and endothelial cells. These cells are produced and destroyed in vivo with a precision that implies the existence of homeostatic mechanisms. The stimuli for endothelial cell proliferation and monocyte production are becoming well characterized. However, the mechanisms involved in eliminating these cells are poorly understood. One mechanism involved in cellular elimination is apoptosis, which can be triggered in some cells by ligation of the Fas molecule. In this report we have investigated whether macrophages and endothelial cells express the Fas molecule, and whether Fas transmits an apoptosis-inducing signal in these cells. We demonstrate that macrophages express Fas and readily undergo apoptosis when cultured with anti-Fas. In contrast, while endothelial cells can express the Fas molecule, Fas ligation is insufficient to induce apoptosis. These results suggest differential regulation of Fas function among cells of the reticuloendothelial system, and imply different mechanisms of homeostasis.     

Fas/Fas ligand system mediates epithelial injury, but not pulmonary host defenses, in response to inhaled bacteria

The Fas/Fas ligand (FasL) system has been implicated in alveolar epithelial cell apoptosis during pulmonary fibrosis and acute respiratory distress syndrome. However, Fas ligation can also lead to cell activation and cytokine production. The goal of this study was to determine the role of the Fas/FasL system in host defenses against Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We administered bacteria by aerosolization into the lungs of Fas-deficient (lpr) mice and wild-type (C57BL/6) mice and measured bacterial clearance at 6 and 12 h. One hour prior to euthanasia, the mice received an intraperitoneal injection of human serum albumin (HSA) for alveolar permeability determinations. At all times after bacterial challenges, the lungs of the lpr mice contained similar or lower numbers of bacteria than those of the C57BL/6 mice. Alveolar permeability changes, as determined by bronchoalveolar lavage fluid HSA concentrations, were less severe in the lpr mice 6 h after the challenges. In response to E. coli, the lpr mice had significantly more polymorphonuclear leukocytes (PMN) and macrophage inflammatory protein 2 in the lungs, whereas histopathologic changes were less severe. In contrast, in response to the gram-positive cocci, the lpr animals had similar or lower numbers of PMN. We conclude that the Fas/FasL system contributes to the development of permeability changes and tissue injury during-gram negative bacterial pneumonia. The Fas/FasL system did not have a major role in the clearance of aerosolized bacteria from the lungs at the bacterial doses tested.     

Critical role for caspases 3 and 8 in neutrophil but not eosinophil apoptosis.

BACKGROUND: Apoptosis is a necessary process to control cell numbers in multicellular organisms. In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation. Here, we studied the role of caspases in neutrophil and eosinophil apoptosis in the presence or absence of granulocyte-macrophage stimulating factor and anti-CD95 monoclonal antibodies, respectively. METHODS: Granulocytes were isolated from human blood using standard protocols. Immunoblot and functional studies with cell-permeable specific peptide inhibitors were performed to analyze caspase involvement. Fas receptor and Fas ligand expression was analyzed by RT-PCR, flow cytometry, and immunoblotting. Cell death was analyzed by ethidium bromide exclusion test. RESULTS: Caspases 3 and 8 are critically involved in the regulation of neutrophil apoptosis in vitro. In contrast, these two caspases did not appear to play a major role in the regulation of eosinophil apoptosis. However, the broad-range caspase inhibitor VAD prevented eosinophil death, indicating that caspases are also involved within the apoptotic machinery of eosinophils. Functional inhibitor studies suggested that caspase 9 is crucial for both caspase 3 and 8 activation, at least in neutrophils. In contrast, spontaneous apoptosis of neutrophils or eosinophils is unlikely to be the consequence of Fas ligand/Fas receptor molecular interactions. CONCLUSION: The data of this study indicate differences in the usage of caspases between neutrophils and eosinophils.     

CD8+ T cells promote inflammation and apoptosis in the liver after sepsis: role of Fas-FasL

Although studies blocking the Fas pathway indicate it can decrease organ damage while improving septic (cecal ligation and puncture, CLP) mouse survival, little is known about how Fas-Fas ligand (FasL) interactions mediate this protection at the tissue level. Here, we report that although Fas expression on splenocytes and hepatocytes is up-regulated by CLP and is inhibited by in vivo short interfering RNA, FasL as well as the frequency of CD8(+) T cells are differentially altered by sepsis in the spleen (no change in FasL, decreased percentage of CD8(+) and CD4(+) T cells) versus the liver (increased FasL expression on CD8(+) T cells and increase in percentage/number). Adoptive transfer of CLP FasL(+/+) versus FasL(-/-) mouse liver CD8(+) T cells to severe combined immunodeficient or RAG1(-/-) recipient mice indicated that these cells could induce inflammation. The FasL-mediated cytotoxic capacity of these septic mouse liver CD8(+) T cells was shown by their ability to damage directly cultured hepatocytes. Finally, although CD8(-/-) mice exhibited a reduction in both CLP-induced liver active caspase-3 staining and blood interleukin-6 levels, only FasL(-/-) (but not CD8(-/-)) protected the septic mouse spleen from increasing apoptosis. Thus, although truncating Fas-FasL signaling ameliorates many untoward effects of sepsis, the pathological mode of action is distinct at the tissue level.     

TIPE2 ameliorates lipopolysaccharide-induced apoptosis and inflammation in acute lung injury

Inflammation Research - Tumour necrosis factor-α-induced protein 8-like 2 (TIPE2) has strong anti-inflammatory properties. However, it is unknown whether increased TIPE2 is protective against...     

Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation.

Epithelial cell injury is the common manifestation of lung injury. Contributing to such injury of epithelial cells is apoptosis. Although apoptosis is part of the normal process of epithelial renewal, in excess it is pathologic. We previously demonstrated the excessive apoptosis of lung epithelial cells and the upregulation of Fas and Fas ligand (FasL) in fibrosing lung diseases. We also showed that inhalation of anti-Fas antibody induced lung injury and fibrosis in mice. Interleukin (IL)-8 is one of the most important cytokines in the pathophysiology of acute lung injury and pulmonary fibrosis. In this study we investigated whether Fas ligation induces IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Bronchiolar epithelial cells underwent apoptosis and also secreted IL-8 in response to tumor necrosis factor (TNF)-alpha or Fas ligation. New gene expression and protein synthesis were not necessary for Fas ligation- and TNF-alpha- mediated apoptosis, but were necessary for IL-8 secretion. We further found that Fas ligation induced activation of nuclear factor-kappa B. We conclude that the Fas/FasL pathway not only mediates apoptosis but also plays a proinflammatory role, and that stimulation of the Fas/FasL pathway in bronchiolar epithelial cells leads to IL-8 production, which may amplify the inflammatory cascade in lung injury and pulmonary fibrosis.     

Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation

Activation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor kappaB activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-gamma, transforming growth factor-ss, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury.     

Repeated episodes of C5a-induced neutrophil influx do not result in pulmonary fibrosis

Multiple reactive oxygen species-induced epithelial injury by glucose, glucose oxidase, and lactoperoxidase instillation in the lung results in a progressive interstitial fibrosis. To test the hypothesis that multiple pulmonary inflammatory responses alone would not result in fibrosis, three sequential inflammatory reactions were produced at weekly intervals in hamster lungs via intratracheal instillation of human recombinant C5a. Numbers of neutrophils and total inflammatory cells in bronchoalveolar lavage (BALF) increased significantly at 24 h after each C5a treatment compared with saline controls. Neutrophils increased by 3-, 33-, and 34-fold compared with the corresponding controls at 24 h after the first, second, and third doses, respectively, but returned to control levels by six days postinstillation. LTB₄ levels increased by 24% and 20% compared with the corresponding controls at 24 h after the first and second doses but were not different from controls at other times. Hydroxyproline levels in treated animals did not differ significantly from control levels throughout the study. Protein levels were significantly increased at 24 h after the second and third doses and six days after the third dose compared with the corresponding controls. Occasional foci of neutrophils in alveolar spaces were observed at 24 h after each dose, but they decreased in frequency after six days. No foci of neutrophils were observed six days after the final dose, although some epithelial degeneration was observed by transmission electron microscopy. Our results indicate that pulmonary inflammation resulting from repeated influx of neutrophils in response to multiple instillations of C5a in the lung does not cause sufficient injury to result in pulmonary fibrosis.     

Cigarette smoke extract induces DNA damage but not apoptosis in human bronchial epithelial cells

Whether DNA damage caused by cigarette smoke leads to repair or apoptosis has not been fully elucidated. The current study demonstrates that cigarette smoke induces single-strand DNA damage in human bronchial epithelial cells. Cigarette smoke also stimulated caspase 3 precursors as well as intact poly (ADP-ribose) polymerase (PARP) production, but did not activate caspase 3 or cleave PARP, while the alkaloid camptothecin did so. Neither apoptosis nor necrosis was induced by cigarette smoke when the insult was removed within a designated time period. In contrast, DNA damage following cigarette smoke exposure was repaired as evidenced by decreasing terminal dUTP-biotin nick-end labeling positivity. The PARP inhibitor, 3-aminobenzamide blocked this repair. Furthermore, cells subjected to DNA damage were able to survive and proliferate clonogenically when changed to smoke-free conditions. These results suggest that cigarette smoke-induced DNA damage in bronchial epithelial cells is not necessarily lethal, and that PARP functions in the repair process. Our data also suggest that the potency of cigarettes as a carcinogen may result from their ability to induce DNA damage while failing to trigger the apoptotic progression permitting survival of cells harboring potentially oncogenic mutations.     

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.     

Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8

Interleukin-15 (IL-15) induces the de novo protein synthesis of intracellular polypeptides and delays neutrophil apoptosis by a mechanism that is still unclear. Herein, we investigated the potential antiapoptotic role of newly synthesized proteins released into the external milieu in IL-15-induced neutrophils. We found that IL-15 induces the de novo synthesis of an approximately 23-kDa protein, representing the predominant protein detected in the milieu, and identified it as IL-1 receptor antagonist (IL-1Ra) by Western blot and immunoprecipitation. We quantified IL-1Ra, IL-1alpha, and IL-1beta concentrations by enzyme-linked immunosorbent assay in intracellular and extracellular fractions from IL-15-induced neutrophils and found that IL-15 does not increase IL-1alpha or IL-1beta production but induces IL-1Ra release. Also, we demonstrated that IL-1Ra does not modulate apoptosis, even at a concentration 250 times greater than that measured in the external milieu. In contrast to granulocyte macrophage-colony stimulating factor, the supernatant harvested from IL-15-induced neutrophils was devoid of antiapoptotic activity. Addition of cycloheximide demonstrates that IL-15 delays apoptosis via de novo synthesis of intracellular proteins and that it increases myeloid cell differentiation factor-1 stability. We demonstrated also that IL-15 decreases the activity of caspase-3 and caspase-8, resulting in an inhibition of vimentin cleavage. Our results indicate that IL-15 can activate an anti-inflammatory loop, based on its ability to induce the synthesis of IL-1Ra by neutrophils. We conclude that IL-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors.     

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells

Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.     

Fas 抗原和caspase-8调控细菌氧化还原蛋白azurin诱导的人骨肉瘤细胞凋亡的实验研究

目的：探讨Fas抗原和caspase-8 在细菌氧化还原蛋白azurin诱导人骨肉瘤细胞凋亡中的调控机制及作用。 方法: 用150 mg/L浓度的azurin处理U2OS 细胞,分别作用6、12、24和48 h。细胞免疫化学法检测Fas 抗原的表达。用荧光试剂盒检测caspase-8 的活性。流式细胞仪检测azurin和抗-Fas-抗体诱导的骨肉瘤细胞凋亡百分率,以及加入caspase-8活性抑制剂IETD-FMK后凋亡百分率的变化。 结果: Azurin诱导U2OS 细胞凋亡后,与对照组比较,Fas 抗原表达强度增加(P<0.01)，caspase-8活性升高(P<0.01)。Fas 抗原表达和caspase-8 活性随着azurin作用时间的延长而逐渐升高,至24 h后达到高峰,然后缓慢下降,但仍显著高于对照组(P<0.01) 。Fas抗原的表达与caspase-8 活性变化呈显著正相关(r=0.873，P<0.01)。表达增强的Fas 抗原具有转导凋亡信号的功能,抗-Fas抗体借此增强azurin诱导U2OS细胞凋亡的作用。Caspase-8 活性抑制剂IETD－FMK能阻断caspase-8 活化而抑制 azurin 或抗-Fas抗体诱导的U2OS细胞凋亡,与抑制剂组比较,细胞凋亡百分率显著差异(P<0.01)。 结论： Fas 依赖途径可能为细菌氧化还原蛋白azurin诱导U2OS 细胞凋亡主要机制之一， caspase-8活性上调可能在凋亡过程中发挥重要作用。     

General,but not myeloid or type II lung epithelial cell,myeloid differentiation factor 88 deficiency abrogates house dust mite induced allergic lung inflammation

Asthma is a highly prevalent chronic allergic inflammatory disease of the airways affecting people worldwide. House dust mite (HDM) is the most common allergen implicated in human allergic asthma. HDM‐induced allergic responses are thought to depend upon activation of pathways involving Toll‐like receptors and their adaptor protein myeloid differentiation factor 88 (MyD88). We sought here to determine the role of MyD88 in myeloid and type II lung epithelial cells in the development of asthma‐like allergic disease using a mouse model. Repeated exposure to HDM caused allergic responses in control mice characterized by influx of eosinophils into the bronchoalveolar space and lung tissue, lung pathology and mucus production and protein leak into bronchoalveolar lavage fluid. All these responses were abrogated in mice with a general deficiency of MyD88 but unaltered in mice with MyD88 deficiency, specifically in myeloid or type II lung epithelial cells. We conclude that cells other than myeloid or type II lung epithelial cells are responsible for MyD88‐dependent HDM‐induced allergic airway inflammation.     

Requirement of caspase-3 for efficient apoptosis induction and caspase-7 activation but not viral replication or cell rounding in cells infected with vesicular stomatitis virus

Infection with vesicular stomatitis virus (VSV), a rhabdovirus and economically significant animal pathogen, was previously demonstrated to induce apoptosis. The mechanism of induction and the role of apoptosis in the VSV-host response have not been completely elucidated. Previous data from our laboratory have suggested that caspase-3 is required for the induction of apoptosis but not viral replication in VSV-infected cells. However, these studies used inhibitors that are selective but not specific for caspase-3. To circumvent this difficulty, we infected both MCF-7 cells which do not express caspases-3 (null), and stable transfectants which express caspase-3 (C3+). When caspase-3 null cells were infected, significant PARP cleavage did not occur, but when C3+ cells were infected, PARP cleavage did occur efficiently. Studies in null and C3+ also suggest that: (1) caspases-3 and -7 are activated sequentially after VSV infection; (2) cell shrinkage and detachment are caspase-3 dependent, but cell rounding is not; and (3) the viral titers were similar between caspase-3 null and C3+ cells suggesting that activation of caspases-3 and -7 are not required for viral replication. Taken together, these results strongly support that the activation of caspase-3 by VSV infection is required for efficient apoptosis induction but not viral replication in vitro. Apoptosis mediated by caspase-3, then, is likely either a host cell response to viral replication or perhaps may be required for in vivo viral replication and spread.     

Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin

Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.     

Expression of Fas and Fas ligand reflects the biological characteristics but not the status of apoptosis of intrahepatic cholangiocellular carcinoma

Although intrahepatic cholangiocellular carcinoma (CCC) is the second most common of hepatic malignancies, there have been few studies evaluating its biological characteristics. We therefore decided to investigate the status of apoptosis and of Fas and Fas ligand expression in this carcinoma. We performed immunohistochemistry for Fas and Fas ligand in 40 CCC cases and evaluated the apoptotic index (AI) by counting the number of morphologically apoptotic cells per 1000 cells. AI was higher in cases with poor differentiation, lymph node metastasis and high Ki-67 labeling index (LI). Fas expression in CCC cells decreased in cases with poor differentiation and high Ki-67 LI. Fas ligand expression in the stromal infiltrating mononuclear cells did not correlate with any clinicopathological features of CCC, but was directly related to Fas expression in CCC cells. Fas ligand was also expressed in CCC cells in 27.5% of the cases and more frequently observed in cases with moderate or poor differentiation and high Ki-67 LI. None of these three parameters correlated with AI. These findings indicate that, in CCC, i) cases showing rapid growth characteristics are less likely to die of Fas-mediated apoptosis and more likely to display counterattack to lymphocytes via Fas ligand expressed by carcinoma cells; ii) stromal mononuclear cells express Fas ligand in response to Fas expression in carcinoma cells; iii) Fas and Fas ligand expression are not related to the status of apoptosis.     

沉默caspase-3基因影响骨髓间充质干细胞的增殖和凋亡

背景：缺血缺氧的心肌微环境导致植入的细胞存活率低。 目的：观察沉默caspase-3基因对大鼠骨髓间充质干细胞增殖和体外缺血缺氧环境下凋亡的影响。 方法：构建靶向caspase-3的shRNA重组慢病毒并转染骨髓间充质干细胞为转基因组,以正常细胞组和空载体组做对照,采用MTS法检测各组细胞增殖情况。建立缺血缺氧模型,real-time PCR和免疫组织化学分别检测缺血缺氧环境各组细胞的caspase-3 mRNA和蛋白表达水平,应用流式细胞术检测不同缺血缺氧时间点(0,6,12,24,48 h)各组细胞的凋亡率。 结果与结论：重组慢病毒成功转染骨髓间充质干细胞,且细胞增殖活性升高(P < 0.05)。缺血缺氧环境下,转基因组细胞caspase-3在mRNA和蛋白表达水平相比对照组下降(P < 0.05)。沉默caspase-3能显著降低骨髓间充质干细胞的凋亡率(P < 0.05),且随着缺血缺氧时间的延长凋亡率缓慢升高。结果提示,沉默caspase-3能加快骨髓间充质干细胞的生长速度和提高在体外缺血缺氧环境下的抗凋亡能力。